Microglia innate immune memory is retained after enforced repopulation [ATAC-seq]

Microglia are crucial for CNS homeostasis and involved in a wide range of neurodegenerative and neuroinflammatory diseases. Systemic inflammation and infections can contribute to neurodegeneration later in life by affecting microglia. Like other innate immune cells, microglia can develop innate immune memory in response to a challenge, altering their response to future stimuli. Innate immune memory can ameliorate or worsen CNS pathology, but its persistence is unclear. Recently, several methods have been developed to stimulate microglia turnover. Here, we investigated whether colony-stimulating factor 1 (CSF1R)-dependent microglia depletion followed by repopulation reversed microglial tolerance to systemic inflammation in mice. Repopulated microglia displayed a reduced expression of homeostatic genes and genes related to mitochondrial respiration and TCA cycle metabolism and an increased expression of immune effector and activation genes. Nonetheless, the blunted inflammatory gene expression induced by the LPS-preconditioning was retained after a depletion-repopulation cycle. Our study highlights the persistence of epigenetic changes underlying microglial tolerance and indicates potential implications of microglia depletion and repopulation therapies on microglia functions. Overall design: We pre-conditioned mice with intraperitoneal LPS (or PBS), transiently depleted microglia with BLZ945, and, after repopulation, assessed microglia gene expression by bulk RNA-seq and chromatin accessibility by bulk ATAC-seq after a secondary LPS (or PBS) challenge. 10-week-old male mice were challenged with 1 mg/kg intraperitoneal (i.p.) LPS from Escherichia coli O111:B4. For unchallenged (PP, PBP) and naïve (PL, PBL) mice, sham injections consisted of the same volume/body mass of Dulbecco's phosphate-buffered saline (DPBS). A week after the first challenge, microglia depletion was induced by colony-stimulating factor 1 receptor (CSF1R) inhibition for five days. Microglia depletion was established with daily administration via oral gavage of the CSF1R inhibitor (CSF1Ri) BLZ945 (or Sotuletinib) at a dosage of 169 mg/kg per day. Control (non-depleted) animals were treated with the same volume/body mass of vehicle containing 20% (w/v) 2-hydroxypropyl-beta-cyclodextrin (HPßCD). Five weeks after the initial challenge, mice were similarly re-challenged with 1 mg/kg LPS or PBS (i.p.) and housed in warming cabinets for three hours before brain tissue collection. Microglia were isolated via FACS, and their chromatin accessibility was assessed by bulk ATAC-seq.